This HIVRAD program represents an integrated approach to exploring the structure and optimizing the immunogenicity of HIV-1 envelope (Env) trimers. The overall goal of the HIVRAD is to make significant advances toward development of an Env-based HIV-1 vaccine that induces a substantial neutralizing antibody response. The role of Project 2 is to structurally characterize proteolytically cleaved, disulfide-stabilized SOSIP gp140 trimers by x-ray crystallography, to determine their three-dimensional structure and to suggest rational improvements in the constructs that would improve stability and immunogenicity. Specific Aim 1: To determine crystal structures of trimeric SOSIP gp140 at <4 A. Using constructs designed in Project 1 and produced in Core B, we will evaluate a series of SOSIP gp140 trimers tailored for crystallizability, both in unliganded form and as complexes with Fabs and/or CD4. We will evaluate Fabs to each of the major neutralization epitopes on Env as well as different CD4-based proteins. We will use state-of-the art robotic screening methods to test the most promsing combinations of Env modifications (deglycosylation, variable loop deletions, etc.), Fabs, and CD4 in order to find suitable conditions for forming diffraction quality crystals. Suitable crystals will be tested again by high throughput automated and robotic methods for diffraction and optimization, and the best crystals will be used for data collection and structure determination by methods such as molecular replacement (MR) and single- (SAD) or multi-wavelength anomalous dispersion (MAD). Specific Aim 2: To improve stability, antigenicity and immunogenicity of the HIV-1 Env trimers by rational design. The crystal structures will be used to enhance the stability and immune properties of the Env trimers. The structural results will be compared and correlated with biological data generated in Project 1 and Core B in order to evaluate the quality and utility of the constructs, and to improve the design of future SOSIP constructs as potential vaccine candidates. RELEVANCE (See instructions): This project is highly relevant for design of an HIV-1 vaccine that would elicit neutralizing antibodies. This project is highly challenging, but currently represents the holy grail in the structural biology of HIV-1. An env trimer structure is required to assess why it is so difficult to mount an effective, potent and broadly neutralizing immune response against primary isolates and across clades.